The focus of research in the Laboratory of Integrative Neuroscience (LIN) is the determination of mechanisms underlying neuromodulation and plasticity and the effects of alcohol and other drugs of abuse on these neuronal functions. Ongoing studies are examining alcohol effects on NMDA and non-NMDA glutamate receptor function. We have used gene-targeted NR2A-/- mice to examine ethanol inhibition of NMDA receptors that contain or lack the NR2A subunit. Ethanol inhibition of NMDARs is enhanced in neurons from NR2A-/- mice, and this effect is more pronounced in cerebellar granule cells than in cortical neurons. We are also undertaking studies to examine alcohol effects on synaptic versus non-synaptic NMDA receptors in autaptic neuronal cultures. We will also examine alcohol effects on NMDAR trafficking between synaptic and non-synaptic pools in this preparation. Recent findings also indicate that ethanol interacts with cyclothiazide actions on AMPA-type glutamate receptors. We have continued studies of synaptic plasticity in dorsal striatum, and are also investigating similar plastic changes in hippocampus and amygdala. We have shown that endocannabinoids, acting as retrograde signals that activate presynaptic CB1 cannabinoid receptors, play a key role in initiation of striatal long-term synaptic depression (LTD). We have accumulated evidence that the endocannabinoids are released from postsynaptic neurons via "backward-transport" involving an anandamide membrane transporter system. We have also examined postnatal development of another endocannabinoid-dependent form of synaptic plasticity, depolarization-induced suppression of inhibition (DSI) in hippocampal CA1 neurons. DSI begins to be expressed at ~P14-P16, and we have evidence that the appearance of DSI is probably due to development of endocannabinoid production mechanisms in CA1 pyramidal neurons. Interestingly, release of endocannabinoids involved in DSI does not appear to involve the transport system implicated in LTD induction. We have also begun to explore the molecular mechanisms involved in endocannabinoid production leading to DSI and LTD in amygdala and striatal neurons using newly-implemented techniques for isolating neurons with attached GABAergic synaptic boutons. Studies using combined HPLC and mass spectrometry have indicated that D2 dopamine receptors, in conjunction with depolarization and glutamate receptor activation, enhance production of the endocannabinoid anandamide in striatum. We have also used these techniques to characterize developmental increases in striatal anandamide levels that appear to be tied to the onset of visual sensory input. Current experiments are exploring the link between enhanced endocannabinoid levels and developmental changes in synaptic plasticity at corticostriatal synapses. Biochemical studies are aimed at understanding the intracellular signals that link receptor activation to induction of plasticity, and determining the mechanisms involved in long-lasting depression and DSI. We have observed that CB1 receptor activation of the ERK-type MAP kinase is mediated predominantly through inhibition of adenylyl cyclase. We hope to determine if this pathway is active in presynaptic terminals. We are also examining effects of CB1 activation on phosphorylation and function of presynaptic vesicle-associated proteins to begin to understand how this receptor may produce lasting alterations in neurotransmitter secretion. The receptors we are examining are targets for drugs of abuse and play prominent roles in neuronal pathways implicated in addiction. In the long-term we are interested in gaining a better understanding of the role of these receptors in drug-seeking and synaptic plasticity during the development of drug tolerance and dependence, as well as addiction. Dr. Andrew Holmes has accepted the position of Acting Chief of the Section on Behavioral Science and Genetics (SBSG). He will initiate studies using gene-targeted mice to examine the molecular basis of alcohol and drug abuse, as well as stress-alcohol interactions. The Section on Structural Biology (SB) that will be set up within the next two years to allow studies of membrane protein structure and alcohol interactions with membrane proteins.